Fine‐tuning the (2S)‐naringenin synthetic pathway using an iterative high‐throughput balancing strategy

Abstract

Metabolic engineering consistently demands to produce the maximum carbon and energy flux to target chemicals. To balance metabolic flux, gene expression levels of artificially synthesized pathways usually fine-tuned using multimodular optimization strategy. However, forward construction is an engineering conundrum because a vast number of possible pathway combinations need to be constructed and analyzed.Here, an iterative high-throughput balancing (IHTB) strategy was established to thoroughly fine-tune the (2S)-naringenin biosynthetic pathway. A series of gradient constitutive promoters from Escherichia coli were randomly cloned upstream of pathway genes, and the resulting library was screened using an ultraviolet spectrophotometry-fluorescence spectrophotometry high-throughput method, which was established based on the interactions between AlCl 3 and (2S)-naringenin. The metabolic flux of the screened high-titer strains was analyzed and iterative rounds of screening were performed based on the analysis results. After several rounds, the metabolic flux of the (2S)-naringenin synthetic pathway was balanced, reaching a final titer of 191.9 mg/L with 29.2 mg/L p-coumaric acid accumulation. Chalcone synthase was speculated to be the rate-limiting enzyme because its expression level was closely related to the production of both (2S)-naringenin and p-coumaric acid. The established IHTB strategy can be used to efficiently balance multigene pathways, which will accelerate the development of efficient recombinant strains. K E Y W O R D S flavonoids, metabolic engineering, modular optimization, promoter Biotechnology and Bioengineering. 2019;116:1392-1404. wileyonlinelibrary.com/journal/bit 1392 |

Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida

“…Heterologous expression of bacterial T3PKSs, including the tetrahydroxynapthalene synthase of S. coelicolor (RppA), has previously been demonstrated in P. putida KT2440 [14,35,36]. Using variants of rppA , we were able to rapidly screen for optimal T3 polyketide production conditions.…”

“…Titers could be improved by modulating this substrate pool. This approach has been successfully used to optimize titers of the T3PKS product naringenin, resulting in titers of 191.9 mg/L [35]. Future work could employ a similar strategy in P. putida to achieve higher titer production of plant T3PKS products.…”

Development of a growth coupled dynamic regulation network balancing malonyl-CoA node to enhance (2S)-naringenin synthesis inE. coli

“…In order to dynamically balance cell growth and malonyl-CoA levels, we attempted to construct a (2S)-naringenin-responsive amplifier through coupling the previously optimized (2S)-naringenin synthetic pathway (Module A) (14) with the abovementioned fatty acid repression module (Module B) ( Fig. 4a).…”

“…In contrast to these approaches, the work present here demonstrates a dynamic optimization coupling that utilizes a growth coupled single cell sensing and regulating DRNs cascade to improve the production of the flavonoid (2S)-naringenin. Specifically, this DRN cascade focuses on malonyl-CoA, a crucial metabolite used as a carbon-chain elongation unit for important compounds such as flavonoids (14), antibiotics (15), and fatty acids (12). However, the availability of intracellular malonyl-CoA is limited and reduces from 0.23 to 0.01 nmol/(mg dry wt) when cells transit from exponential to stationary stage (16).…”

Cell-free prototyping of limonene biosynthesis using cell-free protein synthesis

“…This constraint has often limited cellular approaches to small sets of unique strains, with these sets focused on modifying expression conditions such as ribosome binding site strength Nowroozi et al, 2014), enzyme expression timing , and plasmid architecture (Yang et al, 2016). New developments in parallelized DNA assembly and robotic liquid handling have enabled the testing of 122 plasmid architectures for the 16-gene refactored nitrogen fixation gene cluster from Klebsiella oxytoca (Smanski et al, 2014), the construction of "Marionette" strains for prototyping 243 different expression profiles for lycopene pathway enzymes (Meyer et al, 2019), and the characterization of thousands of ribosome binding site combinations for tuning the production of limonene (Jervis et al, 2019) and naringenin (Zhou et al, 2019). However, these efforts have not been adapted to rapidly characterizing large combinations of enzyme homologs, which can significantly enhance performance (Ma et al, 2011;Tsuruta et al, 2009).…”